The cloned extracellular calcium (CA/2+/o)-sensing receptor (CaR), a G protein-coupled receptor (GPCR, is proven to be a key player in Ca/2+/o homeostasis. Our preliminary studies indicate that homologous and heterologous interactions of the CaR with itself (including with mutant CaRs) and with other GPCRs, respectively, may have important effects on the function of the CaR. Firstly, our previous observation show that an inactivating CaR mutation (R185Q) exhibits a pronounced, dominant negative interaction with the co-transfected wild type receptor, suggesting that one molecule of the Car functionally interacts with another. One of the possible mechanisms for intermolecular "cross-talk" between GPCRs that has been suggested by other researchers is the formation of GPCR heterodimers. Our preliminary studies show that a putative homodimer of the CaR is the major species expressed on the cell surface, suggesting that heterodimerization could be one of the molecular mechanisms underlying negative interaction of the mutant and wild type receptors. Secondly, recent studies show that the CaR is co- localized with a number of other GPCRs, such as the parathyroid hormone (PTH)/parathyroid hormone-related protein (PTHrP) receptor (PTHR) in kidney and the metabotropic glutamate receptors (mGluRs) in brain. Our preliminary studies show that activation of the PTHR by PTH increases the sensitivity of the co-transfected CaR to Ca/2+/o in human embryonic kidney (HEK293) cells. In contrast, one of the mGlur subtypes, mGluRla, blunts the intracellular calcium (Ca/2+/i) responses of the co- transfected CaR to elevated Ca/2+/o or polyamines (CaR agonists) in HEK293 cells. This raises the possibility that the activation of other GPCRs can modulate the sensitivity of the CaR to Ca/2+/o in its tissue specific environment. Our preliminary studies suggest that the CaR may interact with other GPCRs through either direct physical heterodimerization and/or indirect, second messenger-mediated cross- talk. In this proposal, we will utilize the established molecular, biochemical and cell biological tools to determine the underlying mechanisms for these potentially very important receptor-receptor interactions, including those involving one molecule of the CaR with another CaR, the CaR with mGluR1a and the CaR with the PTHR. The specific aims are: First, to test the hypothesis that intermolecular interaction of one molecule of the CaR with another is important for its normal function; second, to understand the mechanism underlying the functional interaction of an inactivating CaR mutant (R185Q) with the co-transfected wild type receptor; third, to examine the molecular basis for functionally negative interaction of mGluR1a, a structurally related GPCR, with the CaR; and finally, to determine the molecular basis for functionally positive effects of the PTHR, a structural unrelated GPCR, on the CaR and, in turn, to examine the possible effects of the CaR on the PTHR.